2008 Progress Report:
Project 4 -- Transport and Fate Particles
EPA Grant Number: R832414C004
Subproject: this is subproject number 004 , established and managed by the Center Director under
grant R832414
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center:
San Joaquin Valley Aerosol Health Effects Research Center (SAHERC)
Center Director:
Wexler, Anthony S.
Title: Project 4
-- Transport and Fate Particles
Investigators:
Wilson, Dennis
,
Louie, Angelique
Institution:
University of California - Davis
EPA Project Officer:
Stacey Katz/Gail Robarge,
Project Period:
October 1, 2005 through
September 30, 2010
Project Period Covered by this Report:
October 1, 2007 through September 30,2008
RFA:
Particulate Matter Research Centers (2004)
Research Category:
Particulate Matter
Description:
Objective:
1) To characterize the time course, tissue distribution, and mechanisms of particulate matter (PM) accumulation in the systemic circulation and target organs. 2) To evaluate the effects of size and surface-fixed charge on this process. 3) To determine how altered lung structure affects systemic particle distribution.
Approach:
Ultrafine particulates are transported into the systemic circulation and target organs by mechanisms that have not been characterized. Key questions include whether particulates in the ultrafine range behave differently from larger particles and whether particle composition, especially surface charge, affects systemic absorption. Experiments in this project will address the mechanisms of transport across the pulmonary epithelial barrier, the time course and target tissues for the distribution of particulates, likely means of intravascular transport, mechanisms of particulate interaction with the vascular wall, and the potential for endothelial cell mediated transport into tissues. We will perform in vivo exposures to particles of varied size and surface-fixed charge. These particulates will be composed of materials traceable by microimaging techniques in real time combined; particles will also be histologically quantitated within target tissues. We also propose in vitro experiments to evaluate potential routes of passive or facilitated transport across epithelial and endothelial cell monolayers. Finally we will determine whether animals with lung structure compromised by postnatal oxidant air pollutant exposure have increased systemic circulation of ultrafine particulates and examine the effect of acute oxidant exposure in adult animals on the transport of particles into the systemic circulation.
Progress Summary:
Specific aim 1: To characterize the time course and distribution of circulating particulates in vivo.
Positron Emission Tomography (PET) is currently being used to determine the deposition and translocation of 78nm 64Cu-labeled amine terminated polystyrene beads in the rat model. Figure 1.1 shows translocation of beads to the heart and mucociliary clearance from the lungs into the GI tract in the rat over a 24 hour time period. Figure 1.2 shows a different translocation pattern of free copper in the rat over 24 hours verifying that the translocation and clearance shown in Figure 1.1 is from intact beads and not free copper. Figure 1.3 shows translocation of beads at 0 and 24 hours when beads are administered intraveneously. This was used to compare to Figure 1.1 which showed heart uptake upon instillation. Intraveneous administration shows primarily liver and spleen uptake. Gamma counting is utilized post vivo for bio-distribution (64Cu and 111In labeled beads). Further verification of label stability was determined in Figure 1.4 with beads incubated in both saline at pH 2 and plasma. These solutions were used to test stability in the stomach and blood, respectively. Over the 48 hour time period the labeled beads remain relatively stable, indicating that the signal shown in Figure 1.1 is mainly from intact beads and not free dissociating copper.
Figure 1.1. 64Cu-labeled amine terminated polystyrene bead translocation in the rat over a 24 hour time period. Beads are shown in lungs, heart and GI tract.
Figure 1.2. Free copper translocation in the rat over a 24 hour time period. Free copper is shown in lungs, kidneys, liver and GI tract.
Figure 1.3. Intravenous administration of beads into the mouse model for comparison to lung bio-distribution. Liver and spleen uptake is shown.
Figure 1.4. Bead stability over 48 hours in saline at pH 2 and plasma.
Future directions for this project include further investigating the mechanism of transport for the heart signal shown in the rat lung instillations. Also, varying surface charge, composition and size of nanoparticles to determine the effects of translocation and overlaying with MRI for anatomical referencing. Blood clearance will be determined for the 78nm beads over the 24 hour time period using gamma counting.
Specific aim 2: To compare the anatomic site of particulate accumulation in tissues with organ distribution as determined by microimaging techniques.
PET imaging has been utilized to determine the deposition pattern for each animal upon instillation. Figure 2.1 shows deposition of polystyrene beads into the lungs vs. stomach. Dynamic imaging has also been implemented to follow translocation of beads over time. Figure 2.2 shows a still image of particles in the trachea and lungs in 2.2a and in lungs in 2.2b. These images were taken over the first hour upon instillation.
Figure 2.1. Verification of deposition of polystyrene beads in the mouse model with lung deposition on the left and stomach deposition on the right.
Figure 2.2. Dynamic imaging of beads in the rat model with the instillation during the scan. 2.2a shows a still of movement in the trachea and lungs and 2.2b shows lung region only. L = lungs, T = trachea.
Future directions for this project include utilizing dynamic imaging to follow translocation of nanoparticles over time in the same animal and utilizing PET imaging in conjunction with gamma counting to determine particulate accumulation in vivo.
Compromised Cardiovascular Animals: Do animals with pre-existing CV disease accumulate nanoparticles at lesions?
The bio-distribution and accumulation of particulates in compromised animals has begun with an atherosclerosis injury model (ApoE) mouse. The instillation material was comprised of 78nm polystyrene-Cu64 and 35nm fluorescently labeled dextran coated particles. PET imaging of the mouse demonstrated deposition of PS particles into lung and Confocal imaging demonstrated transportation of dextran coated particles to other organs. Fluorescence was seen in the descending thoracic aorta (Figure 2.3) and injured right carotid artery (Figure 2.4).
Future directions for this project include synthesizing tri-labeled nanoparticles for PET/MRI/Confocal imaging of a single probe and multimodal imaging (MRI/PET) of particle bio-distribution for both injury and normal animal models.
Specific aim 3: To evaluate potential mechanisms of PM transport across epithelial and endothelial barriers.
Mechanisms of Endothelial and Epithelial transport using synthetic fluorescent and electron dense ultrafine particles.
In previously reported work, we characterized the transport of 30 nm synthetic iron oxide particles across endothelial cell monolayers and demonstrated vesicular transport though caveolar like structures by 4 hours. We further characterized this as vesiculocaveolar transport using fluorescence tagged silica particles with confocal microscopy. We have now extended this work to ask whether similar rates of transport occur in cultured human airway epithelial cells. We found, in contrast to studies with endothelial cells, airway epithelium did not allow transport during a 4 hour incubation period (Figure 3.1 A) compared with extensive transport in endothelium (Figure 3.1B) and furthermore, limited internalization of iron oxide particles occurred despite similar association with cell surfaces (Figure 3.1C). We have recently synthesized Alexafluor 680 conjugated silica particles for use in real time transport studies using deconvolution microscopy. In addition, we have successfully cloned and transfected a GFP caveolin construct that will allow us to co-localize red Alexa 680 labeled PM with green labeled caveoli. Using a new collaboration with the Center for Biophotonics, we propose to follow a time course of particle transport in GFP-caveolin transfected EC using live cells in real time. This technique will then allow inhibitor studies to determine whether this represents an active or passive transport process.
Figure 3.1: Localization of iron oxide particles after 4 hours of incubation with either cultured human airway epithelium (A + C) or aortic endothelium (B). Initial concentration was 10 ug.ml of 30 nm Iron Oxide PM.
Specific aim 4: To characterize the dynamics of interaction between particulates and airways and arterial walls.
We added a new investigator (Dr. Abdul Barakat) to this project to pursue this specific aim. His work has just begun so progress will be reported in the next progress report.
Expected Results:
These experiments use size and surface-fixed charge defined ultrafine particulates to provide baseline information on the time course and extent of their systemic absorption. Understanding the nature of particle transport in blood will be important for recognizing the likelihood and potential mechanisms for interaction with tissues. Combining microimaging of whole animals in real time with quantitative histologic evaluation of tissue distribution should provide insight into the time course and nature of potential biological responses. The high resolution microscopy and reconstruction techniques to be used in these experiments will not only distinguish whether particles move between or through cells and airway or vessels walls but, in combination with inhibitor studies, whether this is an active or passive process, thereby providing insight into the responsible biologic processes.
Journal Articles:
No journal articles submitted with this report: View all 1 publications for this subproject
Supplemental Keywords:
ambient air, ozone, exposure, health effects, human health, metabolism, sensitive populations, infants, children, PAH, metals, oxidants, agriculture, transportation,
,
Air, Health, RFA, Risk Assessments, particulate matter, human health risk, toxicology, epidemiological studies, lung disease, long term exposure
Progress and Final Reports:
2006 Progress Report
2007 Progress Report
Original Abstract
Main Center Abstract and Reports:
R832414
San Joaquin Valley Aerosol Health Effects Research Center (SAHERC)
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R832414C001 Project 1 -- Pulmonary Metabolic Response
R832414C002 Endothelial Cell Responses to PM—In Vitro and In Vivo
R832414C003 Project 3 -- Inhalation Exposure Assessment of San Joaquin Valley Aerosol
R832414C004 Project 4 -- Transport and Fate Particles
R832414C005 Project 5 -- Architecture Development and Particle Deposition
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